Conventional detection reagents for biological assays frequently consist of a binding moiety having specificity for the molecule of interest, conjugated to a moiety with enzymatic or optical properties. To date, these determinations are generally facilitated through the use of radiological, fluorescent or enzymatic tags.
Among methods of interest for analysis, flow cytometry provides the means for simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second, and is routinely used for research and clinical diagnostic applications, including both particle analysis and particle sorting. The analysis of cells is of particular interest. Modern instruments usually have multiple lasers and fluorescence detectors. Increasing the number of lasers and detectors allows for simultaneous analysis of multiple labeled antibodies, and can more precisely identify a target population by their phenotypic markers.
In traditional flow cytometry, fluorescently labeled particles such as live cells, fixed cells, beads, etc. are individually distinguished and separated based on their fluorescence and light scatter characteristics. The phenotype of the particles can be further investigated after they are isolated. Such traditional flow cytometry methods are limited by the number of simultaneous parameters that can be measured on a single particle, and there are problems with overlap of fluorescence emissions during simultaneous measurement; and background fluorescence or enzymatic activity. As the number of simultaneous parameters increases, this spectral overlap severely convolutes analysis impinging on both the accuracy as well as sensitivity of the assay.
In alternative methods of detection, atomic mass spectrometry measurements have been used in conjunction with stable isotope tags of rare elements. Existing elemental tagging capture reagents for use in ICP-MS are based on chelators, such as ethylenediamine tetra-acetic acid (EDTA), tetraazacyclododecane-tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA), for example a maleimide-functionalized polymer of DTPA, with an average length of between 10 and 30 monomers. Such protocols allow conjugation to a typical antibody of 6 or 7 polymers, thereby conjugating an average of 200 tagging isotope atoms per antibody. The sensitivity of this method is directly related to the number of elemental isotope tags per detection reagent molecule. The number of polymers that can be attached is limited to the number of disulfide bonds that can be broken on the immunoglobulin without disrupting its function. The number of metal chelating units that can be conjugated to a detection reagent is also limited because increased numbers can interfere with the detection reagent or induce nonspecific interactions and thus interfering or inducing high background in an assay.
Other existing nanocrystal labeling reagents include luminescent nanocrystals, also known as upconversion nanocrystals, quantum dots, luminescent nanocrystals, and Raman composite organic-inorganic nanoparticles (COINs), all of which were designed for optical or electromagnetic labeling. To achieve useful optical properties, the nanocrystals used in these products contain high-atomic mass dopants, such as cadmium, tellurium, selenium, europium, terbium, or neodymium, and contain undefined mixtures of these rare metals. The high-atomic mass dopants occupy otherwise useful channels of instrument detection. The mixed nature of the elements and their isotopes in these reagents makes them less desirable for atomic mass spectrometry measurement as it would dilute the signal, thus reducing sensitivity, as well as occupy multiple measurement channels per reagent that could otherwise be used as individual reporters.
There is interest in methods of analysis that provide for highly sensitive detection of molecules in biological assays, where multiple parameters can be simultaneously analyzed without signal overlap. The present invention addresses this issue.
Publications
U.S. Pat. No. 7,135,296 Baranov: Elemental analysis of tagged biologically active materials. Winnik et al., J. Anal. At. Spectrom. 2008; 23(4): 463-469, Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry. Winnik et al., Angew. Chem. Int. Ed. 2007; 46 (32): 6111-6114, Polymer-Based Elemental Tags for Sensitive Bioassays. Winnik et al., J. Am. Chem. Soc. 2007; 129(44): 13653-13660, Lanthanide-containing polymer nanoparticles for biological tagging applications: Nonspecific endocytosis and cell adhesion; Thickett et al. (2010) Bio-functional, lanthanide labeled polymer particles by seeded emulsion polymerization and their characterization by novel ICP-MS detection. Journal of Analytical Atomic Spectrometry 25 (3):269-281; Abdelrahman et al. (2010) Lanthanide-Containing Polymer Microspheres by Multiple-Stage Dispersion Polymerization for Highly Multiplexed Bioassays (vol 131, pg 15276, 2009). Journal of the American Chemical Society 132 (7):2465-2465, 2010; Berger et al. (2010) Hybrid nanogels by encapsulation of lanthanide-doped LaF3 nanoparticles as elemental tags for detection by atomic mass spectrometry. Journal of Materials Chemistry 20 (24):5141-5150; Ornatsky et al. (2010) Highly multiparametric analysis by mass cytometry. J Immunol Methods 361 (1-2):1-20; Bandura et al. (2009) Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry. Analytical Chemistry 81:6813-6822